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Membrane trafficking in Drosophila wing and eye
development
Bryan A. Stewart
plex patterned tissue requires an enormous amount
of intercellular signaling. Therefore the Drosophila
eye and wing have long served as developmental models of compartmentalization, cell specification and
patterning. Indeed, founding members of several important signaling pathways that are conserved across
many species, such as Wingless, Notch, and Hedgehog, were first identified by studying genetic mutants
in Drosophila that perturb the development of these
tissues.
In developmental contexts inter-cellular signaling
occurs primarily by the interaction of transmembrane ligands with transmembrane receptors or by
secreted signaling molecules interacting with receptors on cells near to, or far from, the source of the
molecule. Therefore proper intra-cellular trafficking
of secreted molecules, ligands and receptors is essential for proper inter-cellular signaling and thus
development of the tissue.
Recent studies directed at understanding the mechanisms by which intracellular trafficking can effect development of the Drosophila eye and wing have revealed
that subtle perturbations of the trafficking machinery
can produce profound impacts on development of the
adult structures. Collectively these studies reinforce
the idea that regulation of the trafficking pathway may
represent an additional layer of regulation in intracellular signaling pathways. The focus of this paper will
be to review these recent advances in the study of intracellular trafficking in developmental contexts.
It is clear that membrane transport is essential to the
proper sorting and delivery of membrane bound receptors
and ligands, and secreted signaling molecules. Molecular
genetic studies in Drosophila are particularly well suited
to studies of membrane transport in development. The
conservation of cell signaling pathways and membrane
transport molecules between Drosophila and other species
makes the results obtained in these studies of general interest.
In addition, the ability to generate gain- and loss-of-function
genetic mutations of various strengths, and the ability to
generate transgenic flies that direct protein expression to
tissues during development are of particular advantage.
Several recent papers suggest that interesting and novel
roles for membrane transport processes will be uncovered by
studying classically defined membrane transport proteins
in developmental contexts. Together these studies suggest
that regulation of membrane transport may represent an
additional mechanism to regulate the strength of cell–cell
signaling during development.
Key words: exocytosis / endocytosis / SNARE / NSF / p47 /
Hrs / signaling
© 2002 Elsevier Science Ltd. All rights reserved.
Introduction
The adult Drosophila eye and wing arise from primordial tissues known as imaginal discs. In larval stages
the cells of the discs proliferate, and are specified
and patterned. Thus the final adult structure is determined earlier in the fly’s life. The transformation of
the undifferentiated epithelial imaginal disc to a com-
Trans-endocytosis of Notch and Delta in
Drosophila eye development
The early finding1 that Drosophila temperature sensitive mutants of the endocytotic protein dynamin,2, 3
encoded by the shibire locus, give rise to developmental
defects was among the first indications of the importance of membrane trafficking to development. Early
work in the developing Drosophila eye showed that
From the Division of Life Sciences, University of Toronto at Scarborough, 1265 Military Trail, Toronto, Ont., Canada M1C 1A4.
E-mail: [email protected]
© 2002 Elsevier Science Ltd. All rights reserved.
1084–9521 / 02 / $– see front matter
91
B.A. Stewart
internalization of the sevenless tyrosine kinase receptor and its ligand , bride of sevenless, was an important
step in the development of the R7 photoreceptor.4
The importance of endocytosis to the Notch pathway
was demonstrated by Seugnet et al.5 who showed a
genetic interaction between shibire and Notch mutant
alleles in bristle development. Thus, there were several indicators of the importance of endocytosis to
development in general, and to the Notch pathway in
particular.
The Notch signaling pathway, first described in
Drosophila, is a widely conserved signaling cascade
used in cell fate specification.6, 7 The Notch pathway
acts primarily by inhibiting cells within a field from
adopting a particular fate, a process called lateral inhibition. In the ‘core’ Notch pathway ligands, such
as Delta and Serrate in Drosophila, interact with extracellular EGF motifs of the Notch receptor. Activation
of Notch leads to proteolytic cleavage of full-length
Notch and translocation of the Notch intracellular
domain to the nucleus where it acts with Suppressor
of Hairless to activate the transcription of downstream target genes, such as the Enhancer of Split
complex.6, 7
Recent work from Muskavitch and co-workers suggest that an important part of activating the Notch
signaling cascade is endocytosis of the Notch extracellular domain into the ligand expressing cell.8 This
process is called trans-endocytosis. While the dynamics of Delta trafficking have been known for some
time,9 and that improper trafficking of Delta leads
to impairment of Notch signaling,9 the direct influence of endocytosis of Delta upon activation of Notch
signaling was not known.
Parks et al.8 first show that in developing wild-type
eyes that the Notch intracellular domain (NotchICD )
and the Notch extracellular domain (NotchECD ),
though initially expressed in the same cell, are differentially localized during development, implying their
differential trafficking. Indeed NotchECD becomes
localized in cells not previously expressing it. Furthermore, the NotchECD co-localizes with Delta in endocytic vesicles and the incorporation of NotchECD fails
when the temperature sensitive mutant of dynamin,
shits1 , is held at restrictive temperature for short periods of time. Thus, NotchECD is trans-endocytosed into
neighboring cells that express Delta.
Using a genetic allele of Delta, DlRF , that exhibits
trafficking defects they also show that the normal
translocation of NotchECD is disrupted. Finally, another allele, DlCE9 , which is a point mutant in the
third EGF repeat of Delta, fails to initiate trans -
endocytosis of Notch in cell culture assay, and blocks
Notch signaling when expressed in the developing
wing.
These intriguing data show that endocytosis of
NotchECD into the ligand expressing cell is required
for activation of the Notch pathway in the Notch
expressing cell. Several models could account for
this possibility, including: (1) once Notch is bound
to Delta, endocytosis of Delta exerts mechanical
forces on Notch necessary to expose the cleavage
site(s) required to release NotchICD ; or (2) separation of NotchECD from NotchICD is required to
release NotchICD . Consistent with this model is the
observation that secreted Delta, and Delta lacking
its intracellular domain act as suppressors of Notch
signaling.10, 11 In contrast, in at least one instance, an
extracellular fragment of Delta is reported to activate
Notch.12
While it is surprising that endocytosis of part of the
receptor into the ligand expressing cell is necessary
for activation of the signaling pathway, the similarity between trans-endocytosis of Notch and Delta and
the endocytosis of the Boss/Sevenless ligand receptor
complex4 suggests that internalization of large protein complexes may be generally important to developmental cell signaling.
The role of SNARE proteins in signaling at
the Drosophila wing margin
While endocytosis has been recognized in the literature as an important contributor to development,
molecules involved in the exocytotic pathway have
recently come to the fore in developmental contexts. The SNARE (soluble NSF attachment protein
receptors) proteins are a group of three proteins
that are thought to form the minimal machinery
required for fusion of transport vesicles with target membranes.13 The three proteins are from the
VAMP/Synaptobrevin, Syntaxin and SNAP-25 families. Together they can form a very stable complex
that consists of four parallel alpha-helices.14, 15 It is
currently thought that VAMP on the vesicle interacts
with Syntaxin and SNAP-25 on the membrane to form
a trans-membrane complex and that formation of this
‘SNARE complex’ may provide sufficient energy to
cause fusion of lipid bilayers. One indication of the
specific role of SNAREs in a developmental process
was the finding that severe reduction in Syntaxin expression leads to the failure of cellularization of the
Drosophila embryo.16
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Membrane trafficking in Drosophila wing and eye development
It follows that after membrane fusion the SNARE
complex resides in a single membrane and that the
complex needs to be broken apart to allow the proteins to be used in further rounds of membrane fusion.
N-ethylmaleimide sensitive fusion protein (NSF) is an
ATPase that can bind the SNARE complex through
an adaptor called α-SNAP. Upon hydrolysis of ATP by
NSF the SNARE complex breaks apart.17
A dominant negative form of NSF can be engineered by mutating a single amino acid in the ATP
binding region of the ATPase domain.18 To analyze
the role of SNARE dependent membrane trafficking
in Drosophila wing development, a dominant negative
Drosophila NSF2 (dNSF2) isoform was constructed19
and its expression directed to the developing wing
margin using the Gal4–UAS system.20 This resulted in
developmental defects in the wing such that the wings
were notched (Figure 1). Importantly, the wing phenotype was also enhanced by single copy mutations
of the other SNARE genes, synaptobrevin and syntaxin
confirming that other mutations affecting membrane
trafficking can enhance the phenotype. The loss of
wing margin phenotype is reminiscent of phenotypes
obtained with certain mutant alleles of the Notch,
Serrate, and wingless genes. Indeed, there was genetic
enhancement of the wing phenotype when the dNSF2
mutant transgene was introduced into Notch, and
wingless mutant backgrounds indicating the dNSF2
mutant disrupted these signaling pathways.
To confirm this, immunocytochemistry was performed on third instar imaginal wing discs to examine
Wingless and Notch, and some of the downstream
targets of these signaling pathways. Interestingly all
of these markers for wing development were abnormal indicating signaling at the top of the cascade
was disrupted. Direct evidence that both pathways
are affected comes from the observation that both
Wingless and Notch proteins are mislocalized in the
dNSF2 mutant wing discs and that a direct target of
the Notch pathway, the Vestigial boundary enhancer,
is downregulated.
Perhaps the most interesting result to come out of
the study of dNSF2 mutant wing discs is the finding
that they provide an exquisitely sensitive background
that can be used in modifier screens. In this study,
big brain and porcupine, genes previously known to be
involved in Notch and Wingless signaling respectively,
were shown for the first time to be involved in wing
margin development. Thus the partial disruption of
membrane trafficking yields a genetic background
which can be used to screen for molecules involved
in the development of that tissue (Figure 2).
The dNSF2 study focused on wing margin development and the role of Notch and wingless proteins
in that process. An outstanding issue that remains to
be resolved is determining to what degree the dNSF2
mutants effect Notch and Wingless specifically, or is
the effect one common to other signaling molecules.
Figure 1. Dominant negative dNSF2 impairs wing margin development. (A) Wild-type Drosophila wing. (B) Wing from
Drosophila expressing dominant negative dNSF2 along the wing margin. There are clear nicks in the wing. (C) The wing
phenotype is enhanced when the dNSF2 transgene is placed in combination with a genetic mutant of wingless (wg 1–17 )
and with a mutant in (D) Syntaxin (syx L371 ) showing genetic interaction with these two genes. These data were previously
published.9
93
B.A. Stewart
mutant dNSF2 transgene in these cell types may reveal mechanisms of membrane transport important
to development in that tissue.
Role of P47, an α-SNAP homologue, in
Drosophila eye development
The adult Drosophila eye consists of an array of approximately 800 ommatidial units. Each ommatidium
contains eight photoreceptor cells and four cone cells
and a number of support cells. The photoreceptors
are arranged in a trapezoidal array with their apical
surfaces facing the interior of the structure. The rhabdomeres are apical surface membrane expansions of
the photoreceptor cells containing the photosensitive
molecule rhodopsin. Development of the rhabdomere
requires a great amount of membrane trafficking to
generate the expanse of membranous folds.
A very recent study has demonstrated the importance of another ATPase system involved in membrane
fusion for development of the Drosophila eye.23 The
eyes closed (eyc) gene was isolated because of its effects
on rhabdomere morphogenesis. The main phenotype
of the eyes closed mutant is fragmented rhabdomeres
with inappropriate adhesions joining adjacent rhabdomeres. Cloning of the eyes closed gene revealed that
it encodes a homologue of p47, a co-factor that regulates the ATPase, p97.24, 25
Like α-SNAP and NSF, p47 and p9726, 27 act on
SNARE complexes to break apart unproductive
cis-SNARE complexes allowing them to form functional trans-SNARE complexes. The main difference
between the two ATPase molecules and their co-factors
appears to be that α-SNAP and NSF act primarily on
SNARE complexes resulting from heterotypic membrane fusion while p47 and p97 act on those complexes resulting from homotypic membrane fusion.
Exceptions to this generality exist, and in fact, the
NSF and p97 pathways may cooperate in the reassembly of mitotic Golgi fragments24, 28 and both α-SNAP
and p47 can bind to syntaxin-5 containing SNARE
complexes.29
Cellular analysis of photoreceptor development in
eyc mutants revealed that the photoreceptors develop
normally until about 55% of pupal development. At
this time the photoreceptors normally release contacts
between their apical surfaces whereas in the eyc mutants these contacts are abnormally maintained. This
observation was confirmed by immunocytochemical
analysis of adhesive proteins found at this junction,
such as Armadillo and Crumbs, which showed that
Figure 2. Model for the effects of dominant negative
dNSF2 on wing margin development. (A) Notch (N) is normally delivered to the plasma membrane and wingless (w)
is normally secreted from wing margin cells. (B) Sub-lethal
disruption of membrane trafficking leads to reduced Notch
and Wingless transport and the intracellular accumulation
of the proteins. Further reductions in either the signaling
pathway genes or membrane transport genes genetically enhances the phenotype.
For example, there are other secreted and transmembrane modulators of Notch signaling that could be
involved in the phenotypes reported. These include
the secreted proteins Fringe and Braniac, and the
transmembrane modulator Big Brain. Whether these
molecules are similarly affected remains unclear.
The generality of the effects on patterning may
be tested by expressing the mutant dNSF2 in other
domains. This study concentrated on the presumptive wing margin, which is also the dorsal–ventral
boundary of the wing, but it could also be used
to test anterior–posterior patterning. For example,
the major A/P morphogen is the secreted protein
Decapentapalegic21 (Dpp). It seems likely that disruption of Dpp secretion will have profound impacts
on wing development just as disruption of the major
D/V morphogen, Wingless, does. Lastly, the dominant negative dNSF2 transgene is likely to be a useful
tool to study the importance of membrane trafficking in other developmental tissues. As one example,
in Drosophila there is intercellular signaling between
the germ line cells and supporting follicle cells important to oocyte development.22 Expression of the
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Membrane trafficking in Drosophila wing and eye development
these proteins persist at the junctions when they would
normally be cleared.
Molecular analysis of the eyc ORF did not reveal any
point mutations while analysis of the 3 end of the
gene revealed two potential nucleotide substitutions.
Similarly, a transposable P-element that is allelic to
the original eyc mutation is inserted 3 to the ORF
stop codon. Together these data suggest that the eyc1
mutation is one that affects regulation of the gene.
Interestingly, the phenotype of the eyc mutant was
mimicked by misexpression of the wild-type protein
under heat shock control. The severity of the heat
shock induced phenotypes correlated with the number of heat shocks applied throughout development.
Since p97 has previously been implicated in trafficking of vesicles from the endoplasmic reticulum,26, 30
the ER was examined in flies overexpressing eyc. An
abundance of ER was found in those flies with a large
increase in the number of ER stacks observed. This
was further correlated with an increase in the amount
of immature rhodopsin, assayed by Western blot. Together these results suggested a disruption in vesicle
trafficking from the ER may lead to the eyc1 phenotype.
Since eyc encodes a p47 homologue, which regulates the function the p97 ATPase, it seems likely
that the eyc1 phenotype may result from sequestering p97, preventing it from carrying out its normal
functions. Previous in vitro studies on p97 function
revealed the importance of a stoichiometric relationship between p47 and p97,25 thus the overexpression
of p47 will influence p97 function in the developing
Drosophila eye. It also remains possible that the excess
p47 stabilizes unproductive SNARE complexes as can
occur when the yeast α-SNAP homologue, Sec17p is
overexpressed.31
Like the study of dNSF2 dominant negative expression at the wing margin,9 the study of eyc revealed nuanced phenotypes that would not be predicted given
the known function of the protein. Furthermore, it
may be instructive to create transgenic lines with mutations in the p97 ATPase domain to dampen, but not
eliminate, p97 activity.
While on the external surface the eyc1 eyes appear
normal, cellular analysis of the photoreceptors revealed alterations in membrane specialization and
vesicular trafficking that could not have been observed in the complete loss-of-function mutants. It is
presently not clear why overexpression of eyc leads
to a failure of the photoreceptor cells to release their
contacts at their apical surfaces. The failure of the
cells to clear adhesive proteins likely implies the lack
of the machinery normally used in this process. Fur-
ther analysis of the molecular mechanisms underlying
this phenotype will yield interesting new functions for
p47/p97 ATPase activity.
Hrs, a regulator of receptor tyrosine kinase
trafficking
It is clear that manipulating the important components of exocytotic and endocytotic membrane trafficking pathways can have profound impacts upon
development and this suggests that regulation of the
transport pathway itself may be important for development. In support of this idea a recent study shows
that hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a regulatory protein that binds
SNAP-25, one of the SNARE proteins, is an important
regulator of tyrosine kinase receptor trafficking to the
endosome.32
Hrs was first identified as a protein that is phosphorylated in response to hepatocyte growth factor33 and
later identified as a SNAP-25 binding partner by yeast
two-hybrid analysis.34 Hrs has been shown to inhibit
the formation of the SNARE complex in vitro34 and
expression of Hrs in PC12 cells inhibits Ca2+ dependent exocytosis35 suggesting a role for this protein in
exocytosis. However, Hrs is localized to endosomes,
and can also interact with Eps15,36 a protein required
in receptor-mediated endocytosis, further suggesting
that the protein may also be important for endocytosis.
Lloyd et al.32 investigate the function of Hrs by analyzing the Drosophila gene. While there was some localization of the protein to neuromuscular junctions in
wild-type animals, there was no apparent functional
phenotype at this synapse in mutants that live until
pupae. Turning to the garland cells, a tissue with a
high rate of membrane trafficking, they observed
enlargement of endosomes by light microscopy and,
by electron microscopy, failure of the endosomes to
invaginate and form multivesicular bodies.
To examine the developmental consequence of
Hrs loss, they next examined a fly strain in which
the germline cells were homozygous mutant for the
Hrs gene. Embryos lacking maternally derived Hrs
are lethal. In such embryos the activity of two receptor tyrosine kinases, Torso and EGFR, was examined.
Using a combination of immunocytochemistry and
Western blot, it was apparent that there is misregulation of both receptors in Hrs mutants, with reduced
degradation of Torso and EGFR leading to prolonged and spatially disrupted signaling from the two
receptors.
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B.A. Stewart
These results suggest that, in Drosophila, Hrs has a
major role in endosomal trafficking, and in development plays a role in attenuating signals from the tyrosine kinase receptors Torso and EGFR. Hrs has been
proposed to have roles in exocytosis and endocytosis, as well as endosomal trafficking, and it therefore
remains to be determined if, in other developmental
contexts, Hrs may have other important modulatory
roles.
mental contexts, and trying to understand the potential role of membrane transport regulation, will yield
interesting new models on the importance of membrane trafficking to development.
Acknowledgements
I thank D. Ready, T. Lloyd and H. Bellen for providing preprints of their papers prior to publication
and G. Boulianne and T. Lloyd for comments on the
manuscript.
Conclusion
The aim of this paper has been to advance the idea
that membrane transport pathways may have a role in
regulating the strength of intercellular signaling in developmental contexts. While proteins that modulate
some of the signaling pathways directly are well known,
for example Fringe, Numb and Big Brain are modulators of Notch signaling,37 it is becoming evident that
regulated changes in the efficiency of exocytosis or endocytosis may represent another layer of complexity in
regulating the overall strength of cell–cell signaling.
Why might this type of regulation be important?
One possibility is that if all receptors present on
the cell surface are working at peak efficiency, one
way to increase signaling strength is to add more receptors. This is analogous to the finding that rapid,
NSF-dependent, trafficking of neuronal glutamate
receptors is an important mechanism for controlling
the strength of communication between neurons.38
On the other hand, in the case of too much receptor
activity, it may be beneficial to simply remove some
of the cell surface receptors rather than trying to
modulate the function of all of them. Thus regulated
trafficking of receptors and ligands in development
pathways will likely be important for the strength
cell–cell signaling.
Another potentially important scenario for regulated trafficking is that receptors are critical to shaping
the concentration gradients of secreted ligands (see
Cadigan, this issue). It is therefore possible that regulated movement of these receptors may be a means
of controlling the shape of morphogenic concentration gradients. Since molecules such as Wingless are
known to be involved in regulatory feedback loops,39
fine-tuning either secretion of the molecule or insertion of the membrane bound receptor may be an
efficient way of maintaining signaling strength within
a physiological range.
Future studies aimed at bringing together the roles
of membrane transport pathways in specific develop-
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